Phospholipase D Activity of Rat Brain Neuronal Nuclei

Abstract: Phospholipase D activity of rat brain neuronal nuclei, measured with exogenous phosphatidylcholine as substrate, was characterized. The measured activity of neuronal nuclei was at least 36-fold greater than the activity in glia nuclei. The pH optimum was 6.5, and unsaturated but not saturated fatty acids stimulated the enzyme. The optimal concentration of sodium oleate for stimulation of the enzyme activity was 1.2 m M in the presence of 0.75 m M phosphatidylcholine. This phospholipase D activity was cation independent. In the absence of NaF, used as a phosphatidic acid phosphatase inhibitor, the principal product was diglyceride; whereas in the presence of NaF, the principal product was phosphatidic acid. The phospholipase D, in addition to having hydrolytic activity, was able to catalyze a transphosphatidylation reaction. Maximum phosphatidylethanol formation was seen with 0.2–0.3 M ethanol. GTPγS, ATPγS, BeF₂, AIF₃, phosphatidic acid, and phosphatidylethanol inhibited the neuronal nuclei phospholipase D activity. The addition of the cytosolic fraction of brain, liver, kidney, spleen, and heart to the incubation mixtures resulted in inhibition of the phospholipase D activity. Phospholipase D activity was detectable in nuclei prepared from rat kidney, spleen, heart, and liver.     

Endothelin stimulates phosphatidic acid formation in cultured rat mesangial cells: role of a protein kinase C-regulated phospholipase D.

We have previously reported that endothelin-1 stimulates phospholipase C-induced hydrolysis of phosphatidylinositol-4,5-bisphosphate. Other signal transduction pathways that hydrolyze alternative phospholipids through phospholipase D may also mediate endothelin-stimulated cellular responses. We initially evaluated endothelin-dependent generation of 32P-phosphatidic acid as an indirect indication of phospholipase D activity in rat mesangial cells. Endothelin (10(-7) M) induced an elevation of phosphatidic acid that was maximal at 15 min and persisted upward of 60 min. Pretreatment with the diacylglycerol-kinase inhibitor, R59022, did not reduce formation of endothelin-stimulated 32P-phosphatidic acid, demonstrating that the sequential actions of phospholipase C/diacylglycerol kinase do not contribute to endothelin-stimulated phosphatidic acid formation. We next conclusively identified a role for phospholipase D in the generation of phosphatidic acid by assessing the formation of 3H-phosphatidylethanol from 3H-alkyl lyso glycerophosphocholine and exogenous ethanol. Endothelin stimulated 3H-alkyl phosphatidylethanol formation in the presence but not the absence of 0.5% ethanol. Also, endothelin induced a concomitant elevation of 3H-alkyl-phosphatidic acid that was significantly reduced when the cells were exposed to exogenous ethanol, reflecting the formation of phosphatidylethanol. In addition, endothelin stimulated the release of 3H-choline and 3H-ethanolamine, demonstrating that additional phospholipids may serve as substrates for phospholipase D. Phorbol esters and synthetic diglycerides mimicked the effects of endothelin to stimulate phospholipase D and inhibitors of protein kinase C significantly reduced endothelin-stimulated phospholipase D. In addition, endothelin did not stimulate phosphatidylethanol formation in protein kinase C down-regulated cells. The calcium ionophore, ionomycin, did not stimulate phospholipase D and mesangial cells pretreated with BAPTA to chelate cytosolic calcium did not show a diminished endothelin-stimulated phospholipase D. Thus these data demonstrate that mesangial cells possess a protein kinase C-regulated phospholipase D activity that can be stimulated with endothelin.     

Production of phosphatidylethanol by phospholipase D phosphatidyl transferase in intact or dispersed pancreatic islets: evidence for the in situ metabolism of phosphatidylethanol

To determine if phospholipase D is present in intact adult islets, we took advantage of the fact that, in the presence of ethanol, this enzyme generates phosphatidylethanol via transphosphatidylation. Extracts of cells prelabeled with [14C]arachidonate, [14C]myristate, or [14C]stearate were analyzed via three TLC systems; the identify of phosphatidylethanol was further confirmed via incorporation of [14C]ethanol into the same phospholipid bands. The phorbol ester 12-O-tetradecanoylphorbol-13-acetate stimulated phosphatidylethanol (to 603% of basal by 60 min) both in intact adult islets and in dispersed neonatal islet cells. A nonphorbol activator of protein kinase C (mezerein) also stimulated phospholipase D, whereas a phorbol which does not activate protein kinase C (4 alpha-phorbol-12,13-didecanoate) was virtually inactive. The effects of the active phorbol ester or of mezerein were reduced by the protein kinase C inhibitor H-7 and were virtually eliminated by prior down-regulation of that enzyme. In addition, a calcium-selective ionophore (ionomycin) or fluoroaluminate also activated the islet phospholipase D. When accumulation of phosphatidylethanol (labeled with any of three fatty acids) was induced by a preincubation in the presence of ethanol plus agonist, which then were removed, phosphatidylethanol declined by 34-47% over a subsequent 60-min incubation. Thus, while phosphatidylethanol is relatively stable metabolically, it is detectably degraded (a variable overlooked in previous studies). In the absence of ethanol, stimulated islet cells generated phosphatidic acid, although such hydrolysis was less evident than transphosphatidylation. Ethanol provision distinguished phosphatidate formed via phospholipase D (inhibition, via phosphatidylethanol formation) from that due predominantly to phospholipase C (phosphatidate not inhibited). In view of our recent findings that phosphatidic acid (or exogenous phospholipase D) has potent insulinotropic effects, this pathway could play a role in stimulus-secretion coupling; conversely, stimulation of transphosphatidylation at the expense of hydrolysis could contribute to the inhibition of secretion caused by ethanol.     

Phospholipase D activation by platelet-activating factor, leukotriene B4, and formyl-methionyl-leucyl-phenylalanine in rabbit neutrophils. Phospholipase D activation is involved in enzyme release.

Lipid chemoattractants, such as platelet-activating factor and leukotriene B4, as well as the peptide chemoattractant FMLP, were found to stimulate [3H]phosphatidic acid ([3H]PA) formation in 1-O-[3H]octadecyl-lyso platelet-activating factor-labeled rabbit neutrophils. The stimulation of [3H]PA formation appears to result from the activation of phospholipase D (PLD), because in the presence of ethanol, chemoattractant stimulation produced [3H]phosphatidylethanol, the characteristic compound produced by PLD at the expense of [3H]PA formation. The PLD activation by all chemoattractants tested was primed by cytochalasin B and revealed a similar time dependence. However, lipid chemoattractants were less potent as compared with FMLP, and the maximal stimulation by the former was lower than that by the latter. From these results, it is concluded that the mechanism of PLD activation by lipid chemoattractants is similar to, but different from, that by FMLP. Cytochalasin B stimulated degranulation and [3H]PA formation in agonist-stimulated neutrophils, and their stimulations were well correlated. Ethanol inhibited both agonist-stimulated [3H]PA formation and degranulation in a concentration-dependent manner, but the inhibition in degranulation was much less than that in [3H]PA formation. These results suggest that PLD activation is involved in degranulation, but another signaling pathway may also be required for full stimulation of degranulation. When the radiolabeled neutrophils were stimulated by chemoattractants for 5 min, 1,2-[3H]diglyceride was found to accumulate. The accumulation was inhibited by either ethanol or the phosphatidate phosphohydrolase inhibitor propranolol, which indicates that PA produced by PLD can be converted to 1,2-diglyceride by phosphatidate phosphohydrolase. Under these conditions, propranolol did not inhibit degranulation stimulated by chemoattractants. These results indicate that PA produced by PLD is more important than its metabolite diglyceride for the degranulation of rabbit neutrophils.     

Activation of phospholipase D by sphingoid bases in NG108-15 neural-derived cells

Recent studies suggest that signal-dependent formation of phosphatidic acid by phospholipase D-catalyzed hydrolysis of phosphatidylcholine is a novel trans-membrane signaling pathway in mammalian cells. We here demonstrate that sphingosine, as well as some other long chain bases, activates phospholipase D in neural-derived NG108-15 cells. Sphingosine potently stimulated phosphatidic acid and, in the presence of ethanol, phosphatidylethanol formation. (Phosphatidylethanol is a nonphysiological phospholipid which is characteristically produced by phospholipase D in the presence of ethanol.) Elevated phosphatidic acid levels were accompanied by increased phosphatidylinositol and phosphatidylglycerol production and a decrease in diacylglycerol levels. Sphingosine stimulated phospholipase D activity in a time- and concentration-dependent manner. A long aliphatic chain and a free 2-amino group were important structural requirements for the activation of phospholipase D by sphingosine-related molecules. We propose that phospholipase D may constitute an important cellular target for sphingosine action under both physiological and pathological circumstances.     

Angiotensin II induces phosphatidic acid formation in neonatal rat cardiac fibroblasts: Evaluation of the roles of phospholipases C and D

Phosphatidic acid has been proposed to contribute to the mitogenic actions of various growth factors. In³²P-labeled neonatal rat cardiac fibroblasts, 100 nM [Sar¹]angiotensin II was shown to rapidly induce formation of³²P-phosphatidic acid. Levels peaked at 5 min (1.5-fold above control), but were partially sustained over 2 h. Phospholipase D contributed in part to phosphatidic acid formation, as³²P- or³H-phosphatidylethanol was produced when cells labeled with [³²P]H₃PO₄ or 1-O-[1,2-³H]hexadecyl-2-lyso-sn-glycero-3-phosphocholine were stimulated in the presence of 1% ethanol. [Sar¹]angiotensin II-induced phospholipase D activity was transient and mainly mediated through protein kinase C (PKC), since PKC downregulation reduced phosphatidylethanol formation by 68%. Residual activity may have been due to increased intracellular Ca²⁺, as ionomycin also activated phospholipase D in PKC-depleted cells. Phospholipase D did not fully account for [Sar¹]angiotensin II-induced phosphatidic acid: 1) compared to PMA, a potent activator of phospholipase D, [Sar¹]angiotensin II produced more phosphatidic acid relative to phosphatidylethanol, and 2) PKC downregulation did not affect [Sar¹]angiotensin II-induced phosphatidic acid formation. The diacylglycerol kinase inhibitor R59949 depressed [Sar¹]angiotensin II-induced phosphatidic acid formation by only 21%, indicating that activation of a phospholipase C and diacylglycerol kinase also can not account for the bulk of phosphatidic acid. Thus, additional pathways not involving phospholipases C and D, such asde novo synthesis, may contribute to [Sar¹]angiotensin II-induced phosphatidic acid in these cells. Finally, as previously shown for [Sar¹]angiotensin II, phosphatidic acid stimulated mitogen activated protein (MAP) kinase activity. These results suggest that phosphatidic acid may function as an intracellular second messenger of angiotensin II in cardiac fibroblasts and may contribute to the mitogenic action of this hormone on these cells. (Mol Cell Biochem141: 135–143, 1994)Abbreviations DAG diacylglycerol - DMSO dimethyl sulfoxide - lysoPC 1-O-hexadecyl-2-lyso-sn-glycero-3-phosphocholine - NRCF newborn rat cardiac fibroblasts - PA phosphatidic acid - PAPase phosphatidic acid phosphohydrolase - PC phosphatidylcholine - PEt phosphatidylethanol - PI phosphatidylinositol - PL (labeled) phospholipids - PLC phospholipase C - PLD phospholipase D Drs. G. W. Booz and M. M. Taher contributed equally to the work described here.     

Use of [1-14C]oleate labelled autoclaved Escherichia coli as a membranous substrate for measurement of in vitro phospholipase D activity.

Autoclaved Escherichia coli labelled with [1-14C]oleate in the 2-acyl position have been used extensively to measure phospholipase A2 activity in vitro. The present study demonstrates that this membranous substrate is also useful for the measurement of in vitro phospholipase D activity. Phospholipase D from Streptomyces chromofuscus catalyzed the hydrolysis of [1-14C]oleate labelled, autoclaved E. coli optimally at pH 7.0-8.0 to generate [14C]phosphatidic acid in the presence of 5 mM added Ca2+. Other divalent cations would not substitute for Ca2+. Activity was linear with time and protein up to 30% of the hydrolysis of substrate. Phospholipase D activity was stimulated in a dose-dependent manner by the addition of Triton X-100. The activity was increased 5.5-fold with 0.05% Triton, a concentration that totally inhibited hydrolysis of E. coli by human synovial fluid phospholipase A2. Accumulation of [14C]diglyceride was observed after 10 min of incubation. This accumulation was inhibited by NaF (IC50 = 18 microM) or propanolol (IC50 = 180 microM) suggesting the S. chromofuscus phospholipase D was contaminated with phosphatidate phosphohydrolase. Phosphatidic acid released by the action of cabbage phospholipase D was converted to phosphatidylethanol in an ethanol concentration dependent manner. These results demonstrate that [1-14C]oleate labelled, autoclaved E. coli can be used to measure phospholipase D activity by monitoring accumulation of either [14C]phosphatidic acid or [14C]phosphatidylethanol.     

Gonadotropin-releasing hormone activates phospholipase D in ovarian granulosa cells. Possible role in signal transduction

We have investigated the stimulation of phospholipase D activity by the gonadotropin-releasing hormone receptor agonist [D-Ala6, des-Gly10]GnRH N-ethylamide (GnRH-A) in preovulatory, cultured granulosa cells. GnRH-A stimulated up to 10-fold accumulation of phosphatidylethanol, produced by phospholipase D phosphatidyl transferase activity when ethanol acts as the phosphatidyl group acceptor. The effect of GnRH-A was concentration dependent (EC50 = 1 nM) and was inhibited by a specific GnRH receptor antagonist. Low GnRH-A concentrations (less than 10 nM) stimulated also accumulation of phosphatidic acid, but at higher concentrations this response was attenuated. Propranolol, which inhibits phosphatidic acid phosphohydrolase, increased both basal and GnRH-A-stimulated production of phosphatidic acid. A protein kinase C activator, 12-O-tetradecanoylphorbol-13-acetate (TPA, 100 nM), increased up to 30-fold phosphatidylethanol levels. The effects of supramaximal concentrations of GnRH-A (50 nM) and TPA (1 microM) on the accumulation of phosphatidylethanol were additive, suggesting that the two agents may not act via the same mechanism. This is supported by the fact that 1-(5-isoquinolinesulfonyl)-2-methylpiperazine, a protein kinase C inhibitor, inhibited the effect of TPA 50%, but not that of GnRH-A. However, 24 h pretreatment with TPA abolished cellular response to subsequent treatment with either TPA or GnRH-A. The stimulatory action of GnRH on steroidogenesis could be mimicked by elevating endogenous phosphatidic acid levels in granulosa cells. Exogenous phospholipase D (from Streptomyces chromofuscus, 10 IU/ml) significantly increased (2.7-fold) progesterone production by the cells; under the same conditions, GnRH-A and FSH stimulated progesterone production 3- and 2.6-fold, respectively. Similarly, propranolol stimulated progesterone production 2.2-fold. These results suggest that, in granulosa cells, GnRH receptors are coupled to a phospholipase D whose activation may participate in transducing the GnRH signal for accelerated steroidogenesis. Phospholipase D activity can be independently regulated also by protein kinase C. The possible interrelationships between phospholipase D and other phospholipases which may be activated by GnRH in these ovarian cells are discussed.     

Evidence for the calcium-dependent activation of phospholipase D in thrombin-stimulated human erythroleukaemia cells.

Human erythroleukaemia (HEL) cells were exposed to thrombin and other platelet-activating stimuli, and changes in radiolabelled phospholipid metabolism were measured. Thrombin caused a transient fall in PtdInsP and PtdInsP2 levels, accompanied by a rise in diacylglycerol and phosphatidic acid, indicative of a classical phospholipase C/diacylglycerol kinase pathway. However, the rise in phosphatidic acid preceded that of diacylglycerol, which is inconsistent with phospholipase C/diacylglycerol kinase being the sole source of phosphatidic acid. In the presence of ethanol, thrombin and other agonists (platelet-activating factor, adrenaline and ADP, as well as fetal-calf serum) stimulated the appearance of phosphatidylethanol, an indicator of phospholipase D activity. The Ca2+ ionophore A23187 and the protein kinase C activator phorbol myristate acetate (PMA) also elicited phosphatidylethanol formation, although A23187 was at least 5-fold more effective than PMA. Phosphatidylethanol production stimulated by agonists or A23187 was Ca2(+)-dependent, whereas that with PMA was not. These result suggest that phosphatidic acid is generated in agonist-stimulated HEL cells by two routes: phospholipase C/diacylglycerol kinase and phospholipase D. Activation of the HEL-cell phospholipase D in response to agonists may be mediated by a rise in intracellular Ca2+.     

Bradykinin Activates a Phospholipase D that Hydrolyzes Phosphatidylcholine in PC12 Cells

In PC12 pheochromocytoma cells whose phospholipids had been prelabelled with [3H]palmitic acid, bradykinin increased the production of [3H]phosphatidic acid. The increase in [3H]phosphatidic acid occurred within 1-2 min. before the majority of the increase in [3H]diacylglycerol. When the phospholipids were prelabeled with [3H]choline, bradykinin increased the intracellular release of [3H]choline. The production of phosphatidic acid and choline suggests that bradykinin was increasing the activity of phospholipase D. Transphosphatidylation is a unique property of phospholipase D. In cells labeled with [3H]palmitic acid, bradykinin stimulated the transfer of phosphatidyl groups to both ethanol and propanol to form [3H]phosphatidylethanol and [3H]phosphatidylpropanol, respectively. The effect of bradykinin on [3H]phosphatidic acid and [3H]phosphatidylethanol formation was partially dependent on extracellular Ca2+. In cells treated with nerve growth factor, carbachol also increased [3H]phosphatidylethanol formation. To investigate the substrate specificity of phospholipase D, cells were labeled with [14C]stearic acid and [3H]palmitic acid, and then incubated with ethanol in the absence or presence of bradykinin. The 14C/3H ratio of the phosphatidylethanol that accumulated in response to bradykinin was almost identical to the 14C/3H ratio of phosphatidylcholine. The 14C/3H ratio in phosphatidic acid and diacylglycerol was higher than the ratio in phosphatidylcholine. These data provide additional support for the idea that bradykinin activates a phospholipase D that is active against phosphatidylcholine. The hydrolysis of phosphatidylcholine by phospholipase D accounts for only a portion of the phosphatidic acid and diacylglycerol that accumulates in bradykinin-stimulated cells: bradykinin evidently stimulates several pathways of phospholipid metabolism in PC12 cells.     

Formation of phosphatidylethanol in rat brain by phospholipase D

The mechanism of phosphatidyl [14C]ethanol formation was studied in rat brain microsomal fraction. Phospholipase D and base-exchange enzymes were assayed with [14C]ethanol as substrate. Phospholipase D was found to catalyse the formation of phosphatidylethanol. The reaction was dependent on sodium-oleate as activating factor. Phosphatidylethanol formation by phospholipase D has previously only been reported to occur in plant tissues. Stimulation of base-exchange enzymes with calcium in the presence of [14 C]ethanol did not induce any formation of phosphatidylethanol. These findings indicate that phosphatidylethanol formation in ethanol intoxicated rats is catalysed by phospholipase D.     

Regulation of the respiratory burst by cyclic 3',5'-AMP, an association with inhibition of arachidonic acid release.

The mechanism of cAMP regulation of the respiratory burst was studied with HL-60 cells that had been DMSO-differentiated to a neutrophil-like cell. To evaluate the effects of known cAMP concentrations, cells were permeabilized with streptolysin-O. Chemotactic peptide (FMLP)-stimulated NADPH oxidase activity was inhibited by cAMP at concentrations higher than 3 microM. Because intracellular calcium was buffered, inhibitory actions of cAMP were not mediated by modulation of calcium concentration. Effects of cAMP on chemotactic peptide signal transduction mediated by phospholipase C, phospholipase D, and phospholipase A2 were then determined. Neither inositol phosphate generation (phospholipase C) nor phosphatidylethanol generation (phospholipase D activity in presence of 1.6% ethanol) induced by FMLP were significantly affected by cAMP. In contrast, cAMP potently inhibited FMLP-induced arachidonic acid mobilization (phospholipase A2). NADPH oxidase activity induced by exogenous arachidonic acid was not inhibited by cAMP. These results indicate that cAMP-mediated inhibition of arachidonic acid mobilization may be important in regulation of the respiratory burst.     

Resveratrol inhibits the formation of phosphatidic acid and diglyceride in chemotactic peptide- or phorbol ester-stimulated human neutrophils

Resveratrol (trans-3,5,4'-trihydroxystilbene, Res) is a naturally occurring antioxidant found in grape berry skins and red wine. It has anti-inflammatory effects. In this study, we examined the effect of Res on the formation of phosphatidic acid (PA) and diglyceride (DG), in human neutrophils stimulated by formyl-methionyl-leucyl-phenylalanine (fMLP) or by phorbol 12-myristate 13-acetate (PMA). We measured the masses of PA and DG by using a nonradioactive method. Our results showed that Res inhibited the formation of PA in a concentration dependent manner with an IC(50) value of 42.4 and 60.9 microM in fMLP- and PMA-stimulated cells, respectively. Res also suppressed the formation of phosphatidylethanol (PEt), thereby implying inhibition of phospholipase D (PLD) activity. In addition, Res inhibited the formation of both diacylglycerol (DAG) and ether-linked acylglycerol (EAG) induced by fMLP and by PMA. Our results suggest that Res inhibition of PLD activity may contribute to its anti-inflammatory effects.     

Effect of calmodulin antagonists on phospholipase D activity in SH-SY5Y cells

The aim of this study was to investigate the involvement of calmodulin in phospholipase D activation in SH-SY5Y cells. Cells prelabelled with [3H]-palmitic acid were incubated with calmodulin antagonists and/or other compounds. Phosphatidylethanol, a specific marker for phospholipase D activity, and phosphatidic acid were analysed. The calmodulin antagonists, calmidazolium and trifluoperazine, induced an extensive increase in phosphatidylethanol formation, and thus increased basal phospholipase D activity, in a dose- and time-dependent manner. The effect of calmidazolium on carbachol-induced activation of muscarinic receptors was also studied. Calmidazolium did not significantly affect the amount of phosphatidylethanol formed following carbachol addition. However, taking into account the increase in basal activity observed after calmidazolium addition, calmidazolium probably inhibits the muscarinic receptor-induced phospholipase D activation. In addition to phosphatidylethanol, basal phosphatidic acid levels were also increased after calmidazolium and trifluoperazine addition. Incubation with calmidazolium (10 microM) for 10 min induced a two-fold increase in phosphatidic acid. The calmidazolium-induced increase in basal phospholipase D activity was not affected by the protein kinase inhibitors H7 and staurosporine. On the other hand tyrosine kinase inhibitors abolished the calmidazolium-induced activation of phospholipase D. Calmidazolium also induced tyrosine phosphorylation in parallel to the phospholipase D activation. In conclusion, our data indicate that calmodulin antagonists induce phospholipase D activity in SH-SY5Y cells via a tyrosine kinase dependent pathway. This may point to a negative control of phospholipase D by calmodulin although a calmodulin-independent mechanism cannot be excluded. Calmodulin antagonists may be useful tools to further elucidate the mechanisms of phospholipase D regulation.     

Phosphatidylethanol stimulates calcium-dependent cytosolic phospholipase A2 activity of a macrophage cell line (RAW 264.7)

The synthesis of inflammation mediators produced from arachidonic acid is regulated primarily by the cellular concentration of free arachidonic acid. Since intracellular arachidonic acid is almost totally present as phospholipid esters, the concentration of intracellular arachidonic acid is primarily dependent on the balance between the release of arachidonic acid from membrane phospholipids and the uptake of arachidonic acid into membrane phospholipids. Cytosolic phospholipase A(2) is a calciumdependent enzyme that catalyzes the stimulus-coupled hydrolysis of arachidonic acid from membrane phospholipids. Following exposure of macrophages to various foreign or endogenous stimulants, cytosolic phospholipase A(2) is activated. Treatment with these compounds may also stimulate phospholipase D activity, and, in the presence of ethanol, phospholipase D catalyzes the synthesis of phosphatidylethanol. A cell-free system was used to evaluate the effect of phosphatidylethanol on cytosolic phospholipase A(2) activity. Phosphatidylethanol (0.5 microM) added to 1-stearoyl-2-[(3)H]-arachidonoyl-sn-glycero-3-phosphocholine vesicles stimulated cytosolic phospholipase A(2) activity. However, high concentrations (20-100 microM) of phosphatidylethanol inhibited cytosolic phospholipase A(2) activity. Phosphatidic acid, the normal phospholipase D product, also stimulated cytosolic phospholipase A(2) activity at 0.5 microM, but had an inhibitory effect on cytosolic phospholipase A(2) activity at concentrations of 50 and 100 microM. Ethanol (20-200 mM), the precursor of phosphatidylethanol, added directly to the assay did not alter cytosolic phospholipase A(2) activity. These results suggest that phosphatidylethanol alters the physical properties of the substrate, and at lower concentrations of anionic phospholipids the substrate is more susceptible to hydrolysis. However, at high concentrations, phosphatidylethanol either reverses the alterations in physical properties of the substrate or phosphatidylethanol may be competing as the substrate. Both interactions may result in lower cytosolic phospholipase A(2) activity.     

Tumor necrosis factor alpha priming of phospholipase D in human neutrophils. Correlation between phosphatidic acid production and superoxide generation.

Tumor necrosis factor alpha (TNF) primes human neutrophils (PMN) for enhanced superoxide (O2-) production if cells are subsequently stimulated with the chemotactic peptide, n-formyl-Met-Leu-Phe (fMLP). fMLP activates phospholipase D to form phosphatidic acid (PA), and a correlation may exist between PA production and O2- generation in PMN. Therefore, we assessed the ability of TNF to prime phospholipase D activation in PMN stimulated with fMLP. TNF (100 units/ml) pretreatment primed enhanced PA production in PMN challenged with 1 microM fMLP, in the absence of cytochalasin B, as demonstrated by increased production of tritiated PA from PMN label with 1-O-[9',10'-3H]hexadecyl-2-lyso-sn-glycero-3-phosphocholine ([3H]LPAF) and by increased PA mass. PA was formed via activation of phospholipase D and occurred with minimal production of diglycerides. Production of O2- was also enhanced in identically treated cells, and we demonstrated a direct correlation between enhanced PA formation and O2- production. Conversely, ethanol inhibition of PA formation led to a comparable reduction in O2- generation. This report of priming of phospholipase D by physiological agonists is the only natural system where enhanced PA formation has been dissociated from diglyceride formation. Our results suggest a link between PA production and NADPH oxidase activation in human PMN.     

Effect of ethanol on ATP-induced phospholipases C and D and serine base exchange in glioma C6 cells

The effect of extracellular ATP, a nucleotide receptor agonist in the central nervous system, was investigated in glioma C6 cells on the intracellular Ca2+ level and the formation of phosphatidylethanol and phosphatidic acid in the presence and absence of ethanol (150 mM). In the cells prelabeled with [14C]palmitic acid, 100 microM ATP induced both the hydrolysis and the transphosphatidylation reactions leading to the formation of [14C]phosphatidic acid; addition of ethanol generated [14C]phosphatidylethanol. However, ATP-mediated increase in the level of [14C]phosphatidic acid was not inhibited by ethanol. Furthermore, ethanol augmented ATP-induced transient and sustained increase in the intracellular Ca2+ concentration, whereas ethanol alone did not produce any change in the intracellular Ca2+ level. These results indicate that in glioma C6 cells, ATP induces activation of polyphosphoinositide-specific phospholipase C and phospholipase D and that ethanol enhances this effect. In the present investigation we have also shown that long-term (2 days) ethanol treatment, at concentration relevant to chronic alcoholism (100 mM), decreased the incorporation of [14C]serine into phosphatidylserine. Since the effect of ethanol on ATP-induced activities of phospholipase C and phospholipase D and on serine base-exchange in glioma C6 cells differs significantly from that in cultured neuronal cells, these results may contribute to a better understanding of the mechanisms of ethanol action in cells of glial origin.     

Oleate stimulation of diacylglycerol formation from phosphatidylcholine through effects on phospholipase D and phosphatidate phosphohydrolase.

Hydrolysis of exogenous phosphatidylcholine (PtdCho) to 1,2-diacylglycerol by rat liver plasma membranes was stimulated by oleate concentrations as low as 0.1 mM. In the presence of 75 mM ethanol, the fatty acid also enhanced phosphatidylethanol (PtdEtOH) formation from PtdCho. These effects were also observed with linoleate and arachidonate, but not with saturated fatty acids or detergents, and were minimal in microsomes or mitochondria. Release of [3H]choline from exogenous Ptd[3H]Cho was stimulated by oleate, whereas phosphoryl[3H]choline formation was inhibited. Oleate and other unsaturated, but not saturated, fatty acids also stimulated the conversion of exogenous [14C]phosphatidic acid to [14C]diacylglycerol. These data are consistent with stimulatory effects of these fatty acids on both phospholipase D and phosphatidate phosphohydrolase in liver plasma membranes. The stimulatory effect of guanosine 5'-O-[3-thio]triphosphate) (20 microM) on PtdEtOH and diacylglycerol formation from PtdCho was enhanced by low concentrations of oleate. Phospholipase A2 also stimulated PtdEtOH and diacylglycerol formation from exogenous PtdCho. It is proposed that unsaturated fatty acids may play a physiological role in the regulation of diacylglycerol production through activation of phospholipase D and phosphatidate phosphohydrolase.     

Priming of calcium mobilization in human neutrophils by granulocyte-macrophage colony-stimulating factor: Evidence for an involvement of phospholipase D-derived phosphatidic acid

Human neutrophils pre-incubated with granulocyte-macrophage-colony-stimulating factor (GM-CSF) exhibit an enhanced mobilization of calcium in response to secondary stimuli such as chemotactic factors. The mechanisms underlying this priming effect of GM-CSF were examined. It was first demonstrated that the additional calcium mobilized by chemotactic factors in GM-CSF-treated cells was derived from intracellular stores and was associated neither with an increased permeability to calcium nor with production of inositol 1,4,5-trisphosphate. These results indicated that GM-CSF called upon a novel mechanism in order to enhance the mobilization of calcium in human neutrophils. The growth factor has recently been shown to prime phospholipase D leading to an enhanced activation by chemotactic factors and an augmented production of phosphatidic acid. Furthermore the ability of exogenous phosphatidic acid to mobilize calcium in cell types other than neutrophils has been previously demonstrated. Therefore, we examined the potential involvement of phospholipase D in the priming of the calcium response by GM-CSF in human neutrophils. Inhibition of the production of the fMet-Leu-Phe-stimulated production of phosphatidic acid by ethanol or wortmannin had only marginal effects on the concurrent mobilization of calcium. However, the priming of the mobilization of calcium by GM-CSF was greatly decreased in cells treated with either ethanol or wortmannin. These results provide strong support for the hypothesis that the production of phosphatidic acid, which is enhanced in GM-CSF-treated cells, is linked to an increased mobilization of intracellular calcium. These results may have relevance to the mechanism of action of GM-CSF in mature haematopoeitic cells as well to the mitogenic activity of other growth factors.     

Differential regulation of neutrophil phospholipase d activity and degranulation

One of the proposed functions of phosphatidic acid (PA) formation from phospholipase D (PLD) activation in neutrophils is to promote degranulation induced by receptor agonists. The present study shows that the time course and dose response of PA formation and degranulation induced by N-formyl-methionyl-leucyl-phenylalanine (fMLP) differed. PLD activation and degranulation also exhibited different dose response to genistein and epigallocatechin gallate (EGCG), inhibitors of protein tyrosine kinases. Genistein inhibited PLD activity with an IC(50) value of 12.2 microM in fMLP- and 107 microM in phorbol myristate acetate (PMA)-stimulated cells. It required higher concentrations of genistein to inhibit degranulation than to inhibit PLD activity induced by fMLP. EGCG in the range of 40-400 microM had no effect on PLD activity but it inhibited the release of beta-glucuronidase and elastase by fMLP-stimulated cells. These results demonstrate differential regulation of PLD activity and degranulation of primary granules by genistein and EGCG in fMLP-stimulated neutrophils.